GSM7119209: atac_nf_rep1; Gallus gallus; ATAC-seq
NFs were dissected from HH9 embryos as described in the CUT&RUN protocol (n=4 embryos, n=8 NFs) and dissociated in Accumax (Innovative Cell Technologies, AM105) for 30 min at room temperature to achieve a single cell suspension. ATAC-seq was performed as previously described (https://doi.org/10.1038/nmeth.4396). Briefly, dissociated cells were collected by centrifugation at 500 rcf for 5 min in a swinging bucket centrifuge and washed in resuspension buffer containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2. After washing, the cells were resuspended in lysis buffer composed of the resuspension buffer with 0.1% NP-40 (Sigma, I8896), 0.1% Tween-20 (Sigma, P1379), and 0.01% Digitonin (Millipore, 300410). The samples were incubated on ice for 3 min, at which point the lysis buffer was washed out with resuspension buffer containing only 0.1% Tween-20. Upon centrifugation at 500 rcf for 5 min, the supernatant was aspirated, and nuclei were resuspended in 50 ul of transposition mixture containing 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 10% Dimethyl Formamide (Sigma, D4551), 1X PBS, 0.1% Tween-20, 0.01% Digitonin, and 5 ul Tn5 transposase (Illumina, 20034210). Transposition was performed for 1 hour at 37°C in a thermal mixer. The pre-amplification DNA was purified using the Qiagen Minelute Reaction Cleanup Kit (Qiagen, 28204). Library amplification PCR was performed using the Q5® High-Fidelity 2X Master Mix (NEB, M0492S). Library size selection and cleanup was carried out using Ampure XP beads (Beckman Coulter, A63881). Prepared libraries were run on an Agilent 4200 Tapestation with D5000 Reagents and D500 ScreenTape for quality control and final libraires were pooled to equimolar concentrations using the NEB Library Quantification Kit.